人IgE Cε3-Cε4蛋白的表达、复性、纯化及初步鉴定  

The expression, renaturation, purification and preliminary identification of human IgE Cε3-Cε4

作　　者：乔春霞[1] 林周[1] 胡美茹[1] 刘睿[1] 秦卫松[1] 冯健男[1] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所分子免疫室,北京100850

出　　处：《细胞与分子免疫学杂志》2006年第1期64-66,70,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金重大项目资助(No.30490240)

摘　　要：目的获得IgE Fc段的Cε3-Cε4重组蛋白(E34)。方法以RT-PCR技术扩增IgE Cε3-Cε4(E34)cDNA片段,构建原核表达载体。以其转化大肠杆菌BL-21,诱导表达主要以包涵体形式存在的目的蛋白。经复性、纯化后,用Wes-tern blot和ELISA法初步鉴定所获E34蛋白与抗人IgE mAb的结合能力。结果对克隆的E34基因片段测序表明,与已报道的序列相一致。获得的可溶性E34蛋白经SDS-PAGE鉴定显示,其相对分子质量(Mr)同预期的结果相一致。Westernblot和ELISA法进一步证实,E34蛋白能够被鼠抗IgE mAb特异性识别。结论成功地构建了pET28a(+)-E34表达载体,并获得能被抗IgE mAb特异性识别的可溶性蛋白E34,为下一步的工作打下了基础。AIM： To gain recombinant protein Cε3-Cε4 of IgE Fc （E34）. METHODS： We cloned the gene coding human IgE Cε3-Cε4 （E34） and constructed an expression vector pET28a（ ＋ ）-E34. The target protein was expressed as inclusion body in E. coil BL-21. Following renaturation and purification through a CM sephorose FF column, the soluble protein was acquired, and its binding ability to murine anti-hlgE mAb was identified by Western blot and ELISA. RESULTS： The cloned E34 gene was sequenced and proved by SDS-PAGE to be the same as reported sequence. SDS-PAGE analysis showed the relative molecular mass of E34 protein obtained was correct as predicted. Western blot and ELISA data revealed that it owned the epitope of binding to murine anti-hlgE mAb. CONCLUSION： The expression vector pET28a（ ＋ ）-E34 has been successfully constructed and the target protein E34 recognized specifically by murine anti-hIgE mAb is obtained.

关 键 词：IGE Cε3-Cε4 表达 复性 纯化 

分 类 号：R392.11[医药卫生—免疫学]